同时测量新生的转录组和转录组使用4-thiouridine代谢RNA标记和翻译核糖体亲和力净化
Hirotatsu Imai1, Daisuke Utsumi2, Hidetsugu Torihara3
1Department of Investigative Medicine, Graduate School of Medicine, University of the Ryukyus, Nishihara-cho, Okinawa 903-0215, Japan.
Nucleic acids research
|June 28, 2023
概括
研究人员开发了一种新方法,可以同时测量新生的RNA合成和翻译. 这种技术,新生的P-TRAP (nP-TRAP),允许更深入地了解真核生物中的基因表达调节.
科学领域:
- 分子生物学分子生物学
- 基因表达规范 基因表达规范
- 生物化学 生化学
背景情况:
- 基因表达调节对于细胞过程至关重要,如适应和对刺激的反应.
- 同时分析新生的RNA合成和翻译对于理解蛋白质生产至关重要.
- 目前用于同时测量这些过程的基因水平的方法是有限的.
研究的目的:
- 开发一种新的,可靠的方法,在基因层面同时评估新生的RNA合成和翻译.
- 为了使协调转录和翻译调节在真核细胞的分析.
主要方法:
- 开发了一种新的方法,将4-thiouridine (4sU) 代谢RNA标记与翻译核糖体亲和力净化 (TRAP) 结合起来.
- 使用单克隆抗体对保护的核糖体P-茎蛋白进行P-茎介导的TRAP (P-TRAP).
- 在哺乳动物细胞中验证了新生的P-TRAP (nP-TRAP) 方法,特别是在内细胞网膜 (ER) 中的未折叠蛋白质反应 (UPR) 期间.
主要成果:
- P-TRAP技术成功地恢复了内源翻译核糖体,用于在真核生物中进行翻译组分析.
- 在nP-TRAP方法中,在急性ER展开蛋白质反应期间,证明了新生的RNA合成和翻译的动态重编程.
- 开发的方法提供了一个简单而强大的工具,用于分析基因特异性转录-翻译合.
结论:
- 新的新生P-TRAP (nP-TRAP) 方法允许同时测量新生RNA合成和翻译.
- 这种技术有助于研究在转录和转化层面协调基因调节.
- nP-TRAP是一种适用于各种真核生物的多功能工具,用于理解动态基因表达.
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